Phylogenetic Analysis of Giardia lamblia Human Genotypes in Fars Province, Southern Iran

Background: This study is the first phylogenetic genotype analysis of Giardia lamblia in Iran. The main objective was to determine genotyping and identify the sub-assemblages of Giardia lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran, in 2012. Methods: Forty G. lamblia isolates were collected from the patient’s fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, south of Iran. Purification of G. lamblia cysts from fecal samples and DNA extraction were performed using monolayer of sucrose density gradient and Phenol-Chloroform-Isoamylalcohol (PCI) respectively. Semi-nested PCR and sequence analysis were then performed using the primers (GDHeF, GDHiF, and GDHiR) which amplified a 432-bp fragment of Giardia glutamate dehydrogenase (gdh) gene. Phylogenetic analysis was carried out using a neighbor-joining tree composed of the nucleotide sequences of G. lamblia isolates obtained in this study and the known sequences isolates published in GenBank. Results: G. lamblia sub-assemblage AII was the most prevalent genotype with 80% of the cases and 20% of the cases belong to sub-assemblage BIII and BIV based on the DNA sequence of the gdh. G. lamblia isolates at Fars Province were widely distributed within assemblage A cluster (sub-assemblage AII) and the remaining isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV). Conclusion: PCR Sequencing and phylogenetic analysis was a proper molecular method for genotyping and discriminating of the of G. lamblia sub-assemblages in fecal samples, using the glutamate dehydrogenase gene that suggests a human contamination origin of giardiasis.


Introduction
he taxonomy of Giardia has revealed six Giardia species: G. lamblia in mammals, including human; G. agilis in amphibians; G. ardeae and G. psittaci in birds; G. microti and G. muris in rodents (1). G. lamblia (synonym of G. intestinalis and G. duodenalis) is the most common intestinal protozoan parasite of a wide range of mammalian hosts and has a global distribution (2,3). G. lamblia is a species complex with eight major genotypes or assemblages (A-H). Molecular characterizations based on multiple loci are used as the main tool to study genetic variation of different populations of Giardia (4,5). Phylogenetic analysis using the small subunit ribosomal RNA (SSU-rDNA), glutamate dehydrogenase (gdh), beta-giardin (bg) and triose phosphate isomerase (tpi) genes have been used to study genetic variability and relations within assemblages of G. lamblia from different hosts. The gdh locus has been used to successfully genotype isolates of G. lamblia from a range of vertebrate hosts and it allows the discrimination between the various subgenotypes of G. lamblia assemblages (6,7). In this study, glutamate dehydrogenase marker was used in the molecular characterization methods for genotyping and subtyping the isolates of G. lamblia from patients.
G. lamblia is one of the most common intestinal parasites in Iran. The prevalence of giardiasis varies in different parts of Iran (5%-23%) (8). Molecular epidemiology of human giardiasis is still unclear in Iran. Previously, there was no study performed using phylogenetic technique for G. lamblia in Iran. Only little information was available on the G. lamblia genotypes. Three studies based on PCR-RFLP assay, targeting the gdh gene of G. lamblia isolates genotype in Tehran, Shiraz and Isfahan provinces showed that assemblage A was the most prevalent (9)(10)(11). In contrast, two studies based on PCR-RFLP assay, targeting the gdh gene of G. lamblia isolates in Tabriz and in Uremia showed that assemblage B was the most prevalent genotype (12,13).
Molecular studies on G. duodenali isolates and discrimination of genotypes is a useful way to know the transmission route and effective prevention management of giardiasis. The objective of this study was to determine genotyping and identify the sub-assemblages of G. lamblia isolates involved in the transmission of giardiasis in Fars Province, south of Iran. Genotypic characterization of the gdh gene was performed by using genomic DNA directly extracted from human fecal samples to evaluate the potential transmission routes of G. lamblia in this area.

Sample collection
Forty G. lamblia isolates were collected from the patient's fecal samples with gastrointestinal discomfort referred to the health centers and hospitals in Shiraz, Fars Province, South of Iran in 2012. Then, the samples were sent to the Research Laboratory of Intestinal Protozoa in the Department of Parasitology and Mycology in School of Medicine at Shiraz University of Medical Sciences for further examination. Informed consent was taken from the patients and the study was approved by Ethics Committee of the university.

Purification technique
Purification of G. lamblia cysts, which were to be used for DNA extraction, was done using the monolayer sucrose density gradient technique. Single step sucrose gradient with the specific gravity at 0.85 M was performed on positive stool samples in order to concentrate the cysts from fecal samples (14). Aliquots of the purified fecal samples were stored at 4 °C and -20 ºC.

DNA extraction
The genomic DNA of G. lamblia was extracted using the Phenol-Chloroform-Isoamylalcohol (PCI) method with pretreatment by Triton X100 on purified fecal sam-T ples (14). DNA extracts were stored at -20 °C for PCR analysis.

PCR amplification of gdh gene
A semi-nested PCR was performed using three primers known as GDHeF, GDHiF, and GDHiR that amplify a 432-bp fragment of the gdh gene (7)  were included in each round of PCR to validate results. One microliter of PCR product from the primary reaction was added to the secondary PCR containing primers GDHiF and GDHiR. Reactions were visualized in UV on 1.5% agarose gels stained with ethidium bromide.

Sequence analysis of gdh gene
Forty of the PCR products successfully amplified for the gdh locus were sent to First BASE Laboratories (http://www.baseasia.com) for commercial DNA sequencing reactions in both forward and reverse directions using primers GDHiF and GDHiR. Nucleotide sequences were analyzed using the computer program of sequencing scanner software. Multiple alignments and sequence alignment of these sequences were carried out using the BioEdit and the ClustalW MEGA4 programs. The gdh sequences of G. lamblia obtained in this study were aligned with previously published reference sequences of G. lamblia isolates from the GenBank database (Table 1). DNA sequencing of PCR products compared with already known G. lamblia sequences obtained from GenBank using Clus-talW in the MEGA4. The accession numbers obtained for each reference assemblages from GenBank were L40509 (AI), L40510 (AII), AF069059 (BIII), L40508 (BIV), U60984 (C), U60986 (D), U47632 (E), AF069057 (F) and AF069058 (G) (5, 6, and 15). To determine similarities or homologies to known genes in NCBI GenBank sequence database, all obtained nucleotide sequences were submitted to BLAST searches. Sequence representatives for each of the assemblages identified in the studies were submitted to GenBank.

Phylogenetic analysis of gdh gene
Phylogenetic analysis was performed using Neighbor-joining (NJ) method in the MEGA4 program. The evolutionary distance-based analysis was conducted using the Kimura 2parameters model to estimates the distance among assemblages identified in this study. For each calculation, branch reliability was assessed using bootstrap analysis (1000 replicates). A neighbor-joining (NJ) tree composed of the 40-nucleotide sequences of G. lamblia isolates obtained in this study and 33 sequences of G. lamblia isolates of the gdh gene in the GenBank database (Table 1) was reconstructed. The homologous nucleotide sequence of G. ardeae (GenBank accession number AF069060) was used as outgroup.

PCR amplification of gdh gene
The Giardia gdh locus with 432 bp was successfully amplified by semi-nested PCR. Fig. 1 shows a representative gel electrophoresis photograph of the PCR products amplified with gdh primers.

Sequence analysis of the gdh gene
Sequence analysis was performed on 40 PCR products of G. lamblia based on gdh amplification both directions using GDHeF, GDHiF and GDHiR primers.  The genotyping results indicated the presence two main genotypes A and B of G. lamblia. Based on the DNAsequences of the glutamate dehydrogenase, 32 isolates (80%) were detected as assemblage A and 8 isolates (20%) were identical to assemblage B. After performing a BLAST search, analysis revealed all 32 isolates assemblage A sequences were 100% similar to sub-assemblage AII (GenBank Accession number L40510). From 8 isolates as assembalge B, three isolates were 99% similar to sub-assemblage BIII (GenBank Accession number AF069059), one isolate was 99% similar to sub-assemblage BIV (GenBank Accession number L40508) and four isolates were found between sub-assemblage BIII and BIV ( Table 2). Comparison of gdh gene nucleotide substitutions between G. lamblia isolates obtained from this study and GenBank database published reference has shown in Fig. 2.

Phylogenetic analysis of gdh gene
Phylogenetic analysis of gdh DNA sequences was determined to further clarify the relationship of the different genotypes to each other. Fig. 3 shows the Phylogenetic tree based construction on the neighbor-joining method of the gdh sequences from G. lamblia of patients isolates determined in this study and GenBank reference isolates and other G. lamblia assemblages previously published (Table 1).   The phylogenetic tree shows that assemblage A was the most frequent, corresponding with 32 cases (80%). All isolates in cluster A were distributed in subassemblage AII. The second cluster was contained the human assemblage B isolates. Assemblage B was found in 8 cases (20%) of the studied samples. Resulting in assemblage B revealed 2 major lineages designated as assemblages BIII and BIV. Third and fourth were the dog clusters of either assemblage C or D. The fifth cluster was contained the cattle assemblage E. The sixth was contained the cat assemblage F cluster. The seventh was contained the rodent assemblage G cluster. Finally, the eighth cluster was contained the G. ardeae (Fig. 3). Phylogenetic analysis of the gdh gene of G. lamblia isolates from patients provided strong bootstrap support (100%) for the placement of genotypes A and B in separate clusters, indicating the differentiation of the assemblages A and B. Phylogenetic analysis has shown Fars isolates were widely distributed within assemblage A cluster (subassemblage AII) and the remaining Fars isolates were dispersed throughout the assemblage B cluster (sub-assemblage BIII and BIV). No samples were joined in clusters corresponding to assemblages C, D, E and F and G of G. lamblia, also in sub-assemblage AI.

Discussion
Recent progressions on the identification of Giardia assemblages and sub-assemblages have increased tremendously in our understanding of the biology and host-parasite relationship of G. lamblia. In this study, 40 known G. lamblia isolates from patients were used to determine the sub-assemblages that occur in Fars province, Iran. All 40 G. lamblia isolates can be divided into four main groups based on the results obtained from this study. These groups are (i) sub-assemblage AII, (ii) subassemblage BIII, (iii) sub-assemblage BIV and (iv) sub-assemblage BIII/BIV as shown in Table 2. The sequences of gdh for the entire 40 G. lamblia patient isolates were compared to the sequences of known G. lamblia assemblages published by GenBank (Table 1).
In our study, three G. lamblia patient isolates were associated with sub-assemblage BIII and one isolate was associated with subassemblage BIV. Four isolates was situated between sub-assemblage BIII and BIV (Table  2 and Fig. 3). Additionally, Sequencing and phylogenetic analysis of gdh gene sequences from this study and a large set of those which were available from the literature worldwide showed the sequences of the 32 isolates associated with sub-assemblage AII were highly genetically homogenous with 100% homology to published sequences. While the other eight isolates associated with sub-assemblages, BIII and BIV showed variability within the study area.
The results of this study are similar to previous studies (9)(10)(11) in which G. lamblia cysts isolated from human feces were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. In Tehran, the capital of Iran a majority of 87% of the G. lamblia isolates used in the studies were from sub assemblage AII, whereas 7.8% of the isolates were from subassemblage BIII (9). Moreover, In Fars Province, 74.41% isolates were typed as assemblage AII, 17.44% as assemblage BIII, 3.49% as assemblage BIV and 4.66% isolates as mixed assemblages AII and BIV (10). In Isfahan Province in the center of Iran, PCR-RFLP, genotype A group II was detected in 59.7% isolates compared to genotype B that showed 34.32% samples as genotype B Group III and 2.98% sample as genotype B group IV (11). In contrast with our study, assemblage B was predominant in Tabriz and Uremia cities in the northwest of Iran when the gdh gene was targeted by PCR RFLP. In Tabriz analysis, PCR-RFLP revealed that 28 samples (33.3%) were in sub-assemblage AII and 44.4% and 22.2% belonged to subassemblages BIII and BIV, respectively (12). In Uremia, 93.3% of isolates were found to be related to the genotype BIII and 6.7% were related to the subgroup BIV (13).
Current study had focused on the genetic characterization of G. lamblia at the gdh gene using DNA samples from patients' stool samples in Fars Province, Iran. Previous surveys performed on DNA extracted directly from fecal samples in various countries have confirmed that only G. lamblia assemblages A and B are associated with human infections (25). Genotypic analysis showed that only combination of assemblages AII and B are able to infect humans in this area of Iran, suggesting a human contamination origin. Most of our patients with sub-assemblage AII infection had acquired their parasites through the anthroponotic route, mainly involved human-tohuman transmission. Zoonotic transmission may also occur. Reservoir sources of zoonotic transmission were found to be a number of livestock animal species like dairy cattle, companion animals like cats, dogs and wildlife animals (26). Unfortunately, due to limited funding, no animal sample was included in this study, so current result was not enough to demonstrate the role of anthroponotic and zoonotic aspects of giardiasis infections. Further molecular characterization of species, genotypes and subtyping analysis of specimens from both humans and animals and comparisons of various genetic loci are required for identification of the transmission of giardiasis between animals and humans and, understanding of giardiasis transmission.
Geographic distribution of each Giardia assemblage in humans varies in different parts of the world. Our results with predominance of assemblage A was same to studies from Brazil (27)

Conclusion
Human giardiasis is one of the most common intestinal parasitic diseases in Iran, and its molecular epidemiology needs more investigations. PCR sequencing and phylogenetic analysis using the glutamate dehydrogenizes gene are proper molecular methods for G. lamblia genotyping and could discriminate G. lamblia sub-assemblages in fecal samples. The high prevalence of sub-assemblage AII in this study and other similar investigations conducted in Iran reemphasized the importance of applying all the health recommendations. Health education and improvement of sanitation conditions during drinking water and food supply are the most important basic strategies for the control and prevention of anthroponotic transmission of giardiasis in this community. The results could use in the future studies for clinical management and prevention policies.